PROJECT SUMMARY: RNP (ribonucleoprotein) granules are membrane-less compartments in eukaryotic cells which serve as micro-reactors for RNA processing. They form into liquid-like droplets in cells and in vitro, but such property is converted to fibril-like solid state in incurable neurodegenerative diseases, ALS and FTD. One cause of this toxic elements is frequent mutations found in FUS (Fused in Sarcoma), a key protein found in pathologic inclusions of both ALS and FTD patients. In vitro, the mutant FUS protein exhibits accelerated aging into solid fibers whereas the wildtype protein stays in liquid-like form for a longer duration, resembling the disease and normal phenotype, respectively. One outstanding question is the molecular mechanism by which protein and RNA, as a complex drive the assembly of the toxic aggregate. In our published result, we demonstrated that dynamic protein-RNA interaction leads to condensation and tunes the liquid-like property of condensate (PNAS, 2015; Mol Cell, 2016, Science, 2018). We present preliminary data which shows that unlike the wildtype, the ALS/FTD mutants of FUS (i) lose dynamic FUS-RNA interaction (ii) assemble into larger and lower-fluidity droplet in vitro and (iii) higher oligomeric clusters in cells even in the absence of external stress. Building on this exciting finding, we propose to dissect the molecular steps involved in protein-RNA and protein-protein interactions in FUS and ALS/FTD mutants of FUS in Aim 1. A recent breakthrough in the field has been the discovery of disaggregating pathways. Karyopherin-?2 (Kap?2), a nuclear importin was shown to exhibit a robust dissolving activity on FUS-like aggregates in vitro and in cells (four papers in Cell, 2018). In addition, we showed that Ubiquilin-2 (UBQLN2) solubilizes stress granules by disaggregating FUS-like aggregates (PNAS, 2018). We present preliminary data which shows that both Kap?2 and UBQLN2 reverse the defective phenotype of ALS/FTD mutant FUS by recovering dynamic FUS-RNA interaction and normal size of condensates. We will further investigate the dissolution activity of both proteins and potential disaggregating activity of a DDX3 helicase on ALS/FTD mutant FUS in Aim 2. In Aim 3, we will assess the functional consequence of ALS/FTD FUS mutations by testing splicing activity in mammalian cells, measuring cellular oligomerization status by single molecule pull down assay and probing diffusional motion of single FUS-RNP complexes by single molecule tracking in live mammalian cells. The molecular knowledge gained from this study will be invaluable in developing and screening new therapies for both ALS and FTD.